Affiliation: Institute of Molecular Medicine, National Cheng Kung University College of Medicine, Tainan, Taiwan, ROC.

ABSTRACTWhen COS7 fibroblasts and other cells were exposed to UVC irradiation and cold shock at 4°C for 5 min, rapid upregulation and nuclear accumulation of NOS2, p53, WWOX, and TRAF2 occurred in 10-30 min. By time-lapse microscopy, an enlarging gas bubble containing nitric oxide (NO) was formed in the nucleus in each cell that finally popped out to cause "bubbling death". Bubbling occurred effectively at 4 and 22°C, whereas DNA fragmentation was markedly blocked at 4°C. When temperature was increased to 37°C, bubbling was retarded and DNA fragmentation occurred in 1 hr, suggesting that bubbling death is switched to apoptosis with increasing temperatures. Bubbling occurred prior to nuclear uptake of propidium iodide and DAPI stains. Arginine analog Nω-LAME inhibited NO synthase NOS2 and significantly suppressed the bubbling death. Unlike apoptosis, there were no caspase activation and flip-over of membrane phosphatidylserine (PS) during bubbling death. Bubbling death was significantly retarded in Wwox knockout MEF cells, as well as in cells overexpressing TRAF2 and dominant-negative p53. Together, UV/cold shock induces bubbling death at 4°C and the event is switched to apoptosis at 37°C. Presumably, proapoptotic WWOX and p53 block the protective TRAF2 to execute the bubbling death.

Figure 1: UV/cold shock induces nuclear membrane bubbling(A) COS7 cells, with or without expressing EGFP, were exposed to UV irradiation at 480 mJoule/cm2 and then incubated at 4°C for 5 minutes, prior to imaging by time-lapse microscopy at room temperature. Bubble formation (big red and small yellow arrows) from the nucleus is shown. Nuclear EGFP leaked into the gas bubble. See the enlarged pictures are at Supplementary Figure S1 for better resolution. (B) Under similar conditions, COS7 cells were exposed to UV irradiation (480 mJoule/cm2) and then cold shock (4°C for 5 min). The cells were then cultured at 4°C and 37°C, respectively, for indicated times. The extent of bubbling was counted. (C, D) Similarly, COS7 and B16F10 cells were exposed to UV (480 mJoule/cm2) /cold shock (4°C for 5 min) and then incubated at indicated temperatures for 0.5–4 hr. Internucleosomal DNA fragmentation was determined by agarose gel electrophoresis. (E) UV irradiation/cold shock-induced apoptosis is switched to bubbling cell death at low temperature, and vice versa.

Mentions:
When cells were subjected to UV irradiation and/or cold shock and then incubated at 4, 22 or 37°C for indicated durations, formation of a large bubble from the nucleus per cell was counted. UVC was used for all experiments. For example, when COS7 fibroblasts were overexpressed with EGFP (enhanced green fluorescent protein), followed by exposure to UV irradiation and then cold shock at 4°C for 5 min. Time-lapse microscopy was carried out at 22°C or room temperature for 2 hr. Gas was generated in the nucleus and pushed the nuclear and cytoplasmic membranes to pop out a big bubble (Figure 1A; Supplementary Figure S1 for enlarged pictures with better resolution). Nuclear EGFP relocated to the gas bubble with time of incubation. Each cell generated and released one bubble only (Supplementary Figures S1–S3). Neighboring cells, without expressing EGFP, also formed bubbles in a synchronized manner similar to that of the EGFP-expressing cells (Figure 1A). Indeed, upon exposure to UV, all cells underwent bubbling death in a synchronized manner (Supplementary Figure S3). Cold shock for 5 min at 4°C enhances the generation of bubbles. Herein, we designated the event as “bubbling death”.

Figure 1: UV/cold shock induces nuclear membrane bubbling(A) COS7 cells, with or without expressing EGFP, were exposed to UV irradiation at 480 mJoule/cm2 and then incubated at 4°C for 5 minutes, prior to imaging by time-lapse microscopy at room temperature. Bubble formation (big red and small yellow arrows) from the nucleus is shown. Nuclear EGFP leaked into the gas bubble. See the enlarged pictures are at Supplementary Figure S1 for better resolution. (B) Under similar conditions, COS7 cells were exposed to UV irradiation (480 mJoule/cm2) and then cold shock (4°C for 5 min). The cells were then cultured at 4°C and 37°C, respectively, for indicated times. The extent of bubbling was counted. (C, D) Similarly, COS7 and B16F10 cells were exposed to UV (480 mJoule/cm2) /cold shock (4°C for 5 min) and then incubated at indicated temperatures for 0.5–4 hr. Internucleosomal DNA fragmentation was determined by agarose gel electrophoresis. (E) UV irradiation/cold shock-induced apoptosis is switched to bubbling cell death at low temperature, and vice versa.

Mentions:
When cells were subjected to UV irradiation and/or cold shock and then incubated at 4, 22 or 37°C for indicated durations, formation of a large bubble from the nucleus per cell was counted. UVC was used for all experiments. For example, when COS7 fibroblasts were overexpressed with EGFP (enhanced green fluorescent protein), followed by exposure to UV irradiation and then cold shock at 4°C for 5 min. Time-lapse microscopy was carried out at 22°C or room temperature for 2 hr. Gas was generated in the nucleus and pushed the nuclear and cytoplasmic membranes to pop out a big bubble (Figure 1A; Supplementary Figure S1 for enlarged pictures with better resolution). Nuclear EGFP relocated to the gas bubble with time of incubation. Each cell generated and released one bubble only (Supplementary Figures S1–S3). Neighboring cells, without expressing EGFP, also formed bubbles in a synchronized manner similar to that of the EGFP-expressing cells (Figure 1A). Indeed, upon exposure to UV, all cells underwent bubbling death in a synchronized manner (Supplementary Figure S3). Cold shock for 5 min at 4°C enhances the generation of bubbles. Herein, we designated the event as “bubbling death”.

Bottom Line:
Arginine analog Nω-LAME inhibited NO synthase NOS2 and significantly suppressed the bubbling death.Bubbling death was significantly retarded in Wwox knockout MEF cells, as well as in cells overexpressing TRAF2 and dominant-negative p53.Presumably, proapoptotic WWOX and p53 block the protective TRAF2 to execute the bubbling death.

Affiliation:
Institute of Molecular Medicine, National Cheng Kung University College of Medicine, Tainan, Taiwan, ROC.

ABSTRACTWhen COS7 fibroblasts and other cells were exposed to UVC irradiation and cold shock at 4°C for 5 min, rapid upregulation and nuclear accumulation of NOS2, p53, WWOX, and TRAF2 occurred in 10-30 min. By time-lapse microscopy, an enlarging gas bubble containing nitric oxide (NO) was formed in the nucleus in each cell that finally popped out to cause "bubbling death". Bubbling occurred effectively at 4 and 22°C, whereas DNA fragmentation was markedly blocked at 4°C. When temperature was increased to 37°C, bubbling was retarded and DNA fragmentation occurred in 1 hr, suggesting that bubbling death is switched to apoptosis with increasing temperatures. Bubbling occurred prior to nuclear uptake of propidium iodide and DAPI stains. Arginine analog Nω-LAME inhibited NO synthase NOS2 and significantly suppressed the bubbling death. Unlike apoptosis, there were no caspase activation and flip-over of membrane phosphatidylserine (PS) during bubbling death. Bubbling death was significantly retarded in Wwox knockout MEF cells, as well as in cells overexpressing TRAF2 and dominant-negative p53. Together, UV/cold shock induces bubbling death at 4°C and the event is switched to apoptosis at 37°C. Presumably, proapoptotic WWOX and p53 block the protective TRAF2 to execute the bubbling death.